PROJECT SUMMARY ATP, the vital energy source for the cell's diverse metabolic needs, is produced by mitochondria through the process of oxidative phosphorylation (OXPHOS). The survival of many organisms is therefore dependent on the mitochondria's capacity to synthesize ATP. Mitochondrial ribosomes (mitoribosomes) synthesize key components of the OXPHOS system. Mutation and impairment of mitoribosome capacity has been shown to be directly associated with a wide range of pathophysiological neuromuscular conditions, including Leigh syndrome, chronic progressive external ophthalmoplegia, sensorineural deafness, neurological deterioration and infantile cardiomyopathy. Although evolutionarily related to bacterial ribosomes, mitoribosomes contain mitospecific protein features, the functions of which are largely unexplored. The recent publications of cryoEM structures of mitoribosomes have provided powerful and inspiring insights into the organization of these mitospecific elements within the mitoribosome. Armed with this information and combined with the ease of genetic manipulations and lack of dependency on aerobic respiration, yeast is an ideal model system to experimentally address the functional relevance of mitospecific features of the mitoribosome. Recent clinical studies have demonstrated that mutation of the mitoribosomal MRPL44 (mL44 family) has been directly linked to childhood- onset hypertrophic cardiomyopathy and a progressive multisystem disease with neurological and neuro- ophthalmological impairment. This disease is caused by mutation of residue Leu156(Arg) in a structurally conserved region of the MRPL44 protein. The cryoEM structural studies indicate that MRPL44 is a conserved mitospecific protein located at the membrane protuberance region of the large ribosomal subunit, proposed to be critical for the membrane tethering of mitoribosomes. Membrane tethering is vital for mitoribosome assembly and the co-translational membrane insertion of proteins being synthesized by the mitoribosomes. The goal of this R15 application is to perform a feasibility study to explore the use of the yeast Saccharomyces cerevisiae (S. cerevisiae) as a genetic and biochemically tractable organism for modeling human mitochondrial diseases caused by defects in the mitoribosomes, and specifically in the mL44 protein. We aim to functionally characterize the yeast mL44 protein, MrpL3 and its partner proteins MrpL15/mL57 and to specifically to model the MRPL44 Leu156(Arg) disease in this organism. The proposed pilot project represents the first experimental exploration into the membrane protuberance region of the mitoribosome and its functional significance for the coupling of translation to the downstream membrane insertion and OXPHOS assembly events.